Enzymes represent a significant percentage of drug targets for the treatment of life threatening disease as exemplified by the effective management of AIDS with cocktails of HIV protease and reverse transcriptase inhibitors and the widespread treatment of cardiovascular disease with statins, angiotensin converting enzyme inhibitors, and most recently, renin inhibitors. Indeed, an analysis of the top 20 marketed drugs established that over one third were enzyme inhibitors. The two primary strategies for the development of small molecule enzyme inhibitors are (1) high throughput screening (HTS) of large compound collections, and (2) conversion of natural substrates to mechanism-based inhibitors. While both strategies have successfully been applied to a number of enzyme targets, for several classes of enzymes, neither approach has proven to be particularly effective. For these enzyme classes HTS has resulted in low hit rates and/or a high rate of false positives (compounds that are incorrectly identified as inhibitors) and natural substrate-based methods have resulted in non-drug like inhibitors with poor efficacy in vivo. A powerful new method for small molecule inhibitor discovery called Substrate Activity Screening (SAS) is proposed. The SAS method, which is the first substrate-based method for fragment discovery and optimization, consists of three steps: (1) a diverse library of low molecular weight substrates is screened against the enzyme target to identify hit fragments, (2) the identified fragments are rapidly optimized by subsequent rounds of analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct incorporation of mechanism-based inhibitor pharmacophores. Screening for substrate as opposed to ligand fragments has two significant advantages. Because the assay requires productive substrate binding and turnover, false positives often seen in traditional high-throughput inhibitor screens are eliminated. Secondly, catalytic substrate turnover results in signal amplification enabling the identification of very weakly active lead fragments. The SAS method will be developed for cysteine proteases and tyrosine protein phosphatases, two large enzyme classes that encompass many important targets for the treatment of life-threatening disease and for which inhibitor development by traditional methods has been challenging. The SAS method will be demonstrated by the development of potent small molecule inhibitors to: (1) the cysteine protease cruzain, which is a validated target for the treatment of Chagas' disease, (2) the two essential cysteine proteases encoded by Trypanosoma brucei for the treatment of African sleeping sickness, (3) the dipeptidyl peptidase DPAP3 encoded by Plasmodium falciparum that recently was established as an extremely promising target for the treatment of malaria, and (4) the two phosphatases encoded by the Mycobacterium tuberculosis, PtpA and PtpB, both implicated as important new therapeutic targets for the treatment of tuberculosis. PUBLIC HEALTH RELEVANCE: Inhibitors of enzyme represent greater than 30% of all therapeutic agents. A new method will be developed that will enable the rapid identification of new, potent and drug like enzyme inhibitors. The method will be demonstrated by developing enzyme inhibitors that could serve as leads for the development of drugs to treat the life-threatening neglected diseases malaria, tuberculosis, Chagas' disease and African sleeping sickness.